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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/05—Dipeptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
  • A61K31/27—Esters, e.g. nitroglycerine, selenocyanates of carbamic or thiocarbamic acids, meprobamate, carbachol, neostigmine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/4164—1,3-Diazoles
  • A61K31/4178—1,3-Diazoles not condensed 1,3-diazoles and containing further heterocyclic rings, e.g. pilocarpine, nitrofurantoin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/005—Enzyme inhibitors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
  • A61P25/16—Anti-Parkinson drugs
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

• DR death domain receptors
• necrostatin can be a Nec-1 related compound of Formula I-E:
• Ri 4 is acyl, acetyl, alkyl, halogen, amino, acylamino, nitro, -SRn, - (Rn)2, or -ORn; the bond indicated by (a) can be a single or double bond; and
• the bond indicated by (b) can be a single or double bond.
• necrostatin can be a Nec-2 related compound of Formula IIA:
• R 3 , R4, R 8 , and R9 are Ci-C 6 alkoxyl.
• R 8 and R9 each represent independently hydrogen, Ci-C 6 alkyl, heteroalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;
• necrostatin can be a Nec-4 related compound of Formula IV:
• R2 and R3 each represent independently for each occurrence hydrogen or methyl
• R4 represents independently for each occurrence halogen, hydrogen, Ci-C 6 alkyl, C 2 - C 6 alkenyl, or C2-C 4 alkynyl;
• R 5 is Ci-C 4 alkyl
• R6 is hydrogen, halogen, or -CN
• A is phenylene or a 5-6 membered heteroarylene
• Ri is Ci-C 6 alkyl, halogen, hydroxyl, d-C 6 alkoxyl, -N(R4) 2 , -C(0)R4, CO2R4, or
• R4 represents independently for each occurrence hydrogen, Ci-C 6 alkyl, aryl, or aralkyl
• B is a 5-6 membered heterocyclic or carbocylic ring
• p 0, 1, or 2.
• necrostatin can be a Nec-7 related compound of Formula VIII:
• each Z 1 and Z 2 is selected, independently, from O, S, or NR Z1 ;
• Xi and X 2 are, independently, N or CR 4 ;
• R 1 is selected from H, halogen, optionally substituted Ci- 6 alkyl, or optionally substituted
• a necrostatin e.g., necrostatin- 1 or necrostatin-4
• a pan-caspase inhibitor e.g., ZVAD or IDN-6556
• the combination treatment of a necrostatin and a pan-caspase inhibitor preserves neuron viability and promotes nerve function in a CNS neuron following CNS injury.
• CNS disorders characterized by impaired or failing axon growth or axon degeneration may arise from CNS neuron injury (e.g., trauma, surgery, nerve compression, nerve contusion, nerve transection, neurotoxicity, or other physical injury to the brain or spinal cord) or neurodegenerative CNS disease, wherein a symptom of the disorder is axon degeneration (e.g., Alzheimer's disease, amyotropic lateral sclerosis (ALS/Lou Gehrig's Disease), Parkinson's disease, multiple sclerosis, diabetic neuropathy, polyglutamine (polyQ) diseases, and stroke, Fahr disease, Menke's disease, Wilson's disease, cerebral ischemia, prion disorder (e.g., Creutzfeldt-Jakob disease).
• CNS neuron injury e.g., trauma, surgery, nerve compression, nerve contusion, nerve transection, neurotoxicity, or other physical injury to the brain or spinal cord
• a symptom of the disorder is axon degeneration (e.g., Alzheimer
• ZVAD also known as Z-VAD, Benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone and carbobenzoxy-valyl-alanyl-aspartyl-[0-methyl]- fluoromethylketone
• IDN-6556 also known as "PF -3 ,491,390" available from Conatus Pharmaceuticals, Inc.
• necrostatin As used herein, the term "necrostatin” or “nec” is understood to mean an inhibitor of caspase-independent cell death or necroptosis.
• exemplary necrostatins include necrostatin-1 ("Nec-1"), necrostatin-2 ("Nec-2”), necrostatin-3 (“Nec-3"), necrostatin-4 ("Nec-4"), necrostatin-5 (“Nec-5”) and necrostatin-7 (“Nec-7").
• R2 is hydrogen or Ci-C 6 alkyl.
• X is O.
• Ri is hydrogen or halogen (such as chlorine).
• R2 is a methyl or ethyl.
• Ri is hydrogen or CI, and R2 is a methyl.
• the necrostatin is a Nec-1 related compound of Formula I- A, shown below:
• R is H, alkyl, alkoxyl, or a halogen (for example, F, CI, Br or I) and R2 is H or an alkyl.
• Ri is H or CI.
• R2 is a methyl or ethyl.
• Ri is H or CI, and R2 is a methyl.
• the necrostatin is a Nec-1 related compound of Formula I-C, shown below:
• the necrostatin is a Nec-1 related compound of Formula I-D, shown below:
• necrostatin is a Nec-1 related compound of Formula I-G, shown below:
• Ri, R2, R3, R4, R5, R6, R7, Re, R9, and Rio each represent independently hydrogen, acyl, acetyl, alkyl, halogen, amino, Ci-C6alkoxyl, nitro, -C(0)Ri2, -C(S)Ri2, -C(0)ORi2,
• Ri 4 is acyl, acetyl, alkyl, halogen, amino, acylamino, nitro, -SRn, -N(Rn)2, or -ORn; the bond indicated by (a) can be a single or double bond; and the bond indicated by (b) can be a single or double bond.
• X is -C(O)-.
• Ri, R2, R5, R6, R7, and Rio each represent independently hydrogen, acyl, alkyl, halogen, or amino.
• R 3 , R4, Rs, and R 9 are Ci-C 6 alkoxyl.
• the bond indicated by (a) is a double bond; and the bond indicated by (b) is a double bond.
• Nec-2 related compounds described above can be prepared based on synthetic procedures described in the literature, such as in International Patent Application Publication No. WO 2007/075772, which is hereby incorporated by reference.
• R 6 is -C(0)R 8 , -C(S)R 8 , -C(0)OR 8 , -C(0)NR 8 R 9 , -C(S)NR 8 R 9 , -C(NH)R 8 , or -S(0 2 )R 8 ;
• R2 and R3 each represent independently for each occurrence hydrogen or methyl
• R4 represents independently for each occurrence halogen, hydrogen, Ci-C 6 alkyl, C 2 - C 6 alkenyl, or C2-C 4 alkynyl;
• R 5 is Ci-C 4 alkyl
• R6 is hydrogen, halogen, or -CN
• R7 is hydrogen or Ci-C 4 alkyl
• Y is N or -C(Rio)-
• Z is S or O
• Ri is . In certain other embodiments, Ri is . In certain embodiments, R2 is hydrogen. In certain embodiments, R 3 is methyl. In certain other embodiments, R 3 is hydrogen. In certain embodiments, R4 is halogen, such as fluorine or chlorine. In certain embodiments, R4 is halogen. In certain embodiments, R5 is methyl or ethyl. In certain embodiments, R6 is -CN. In certain embodiments, A is phenylene. In certain embodiments, X is N. In certain embodiments, Y is N. In certain embodiments, Z is S. In certain embodiments, A is phenylene. In certain embodiments, Ri is Ci-C 6 alkyl, such as methyl. In certain embodiments, m is 1. In certain embodiments, n is 2.
• the necrostatin is a Nec-4 related compound of Formula
• IV-A a pharmaceutically acceptable salt thereof.
• Nec-4 related compounds described above can be prepared based on synthetic procedures described in the literature, such as in Teng et al, (2007) BiooRG MED CHEM LETT, 17: 6836-6840; and Teng et al, (2008) BiooRG MED CHEM LETT, 18: 3219-3223, both of which are incorporated herein by reference.
• the necrostatin is a Nec-5 related compound of Formula V:
• Ri is Ci-C 6 alkyl, halogen, hydroxyl, d-C 6 alkoxyl, - ⁇ (3 ⁇ 44) 2 , -C(0)R4, CO2R4, or
• R 3 is -Ci-C 6 alkylene-CN, -CN, Ci-C 6 alkyl, or C 2 -C 6 alkenyl;
• B is a 5-6 membered heterocyclic or carbocylic ring
• n and p each represent independently 0, 1, or 2.
• R 2 In certain embodiments, R3 is -Ci-C 6 alkylene-CN, such as -CH 2 -CN. In certain embodiments, R4 represents independently for each occurrence hydrogen or Ci-C 6 alkyl. In certain embodiments, R5 represents independently for each occurrence Ci-C 6 alkyl, halogen, hydroxyl, or Ci- C 6 alkoxyl. In certain embodiments, B is a 5-6 membered heterocyclic ring. In certain embodiments, n is 0. In certain embodiments, p is 0.
• the necrostatin is a Nec-5 related compound of Formula V-A:
• Ri is Ci-C 6 alkyl, halogen, hydroxyl, Ci-C 6 alkoxyl, or -N(R 4 ) 2 ;
• R 3 is -Ci-C 6 alkylene-CN
• R4 represents independently for each occurrence hydrogen, Ci-C 6 alkyl, aryl, or aralkyl
• R5 represents independently for each occurrence Q-Cealkyl, halogen, hydroxyl, Ci- C 6 alkoxyl, - ⁇ ) 2 , -C(0)R4, CO2R4, or C(0)N(R4) 2 ;
• B is a 5-6 membered heterocyclic or carbocylic ring
• n and p each represent independently 0, 1, or 2.
• the necrostatin is a Nec-7 related compound of Formula VII:
• A is a 5-6 membered heterocyclic ring
• p 0, 1, or 2.
• R3 is hydrogen.
• R4 is .
• R5 is halogen, Ci-C 6 alkyl, hydroxyl, Ci-C 6 alkoxyl, or -N(R 7 ) 2 .
• R5 is halogen, such as fluorine or chlorine.
• p is 0.
• R4 is , such as .
• the Nec-7 related compound is or a
• Nec-7 related compounds described above can be prepared based on synthetic procedures described in the literature, such as in Zheng et ah, in BIOORG MED CHEM LETT, 2008, vol. 18, 4932-4935, which is incorporated herein by reference.
• the necrostatin is a Nec-7 related compound of Formula VIII:
• each X 1 , X 2 , X 3 , X 4 , X 5 , and X 6 is selected, independently, from N or CR X1 ;
• each Y 1 , Y 2 , and Y 3 is selected, independently, from O, S, NR Y1 , or CR Y2 R Y3 ;
• each Z 1 and Z 2 is selected, independently, from O, S, or NR Z1 ;
• R 1 is H
• X 1 , X 2 , and X 4 are each CH
• X 3 , X 5 , and X 6 are each N
• Y 1 and Y 3 are each S
• Y 2 is NH
• Z 1 is NH
• Z 2 is O
• R 2 is not 4-fluorophenyl
• X 1 , X 2 , X 4 , X 5 , R 1 , Y 2 , and R Z1 are as defined for Formula (VIII);
• R and R , R and R , or R and R combine to form an optionally substituted cycloalkyl or an optionally substituted heterocyclyl;
• R 10 and R 11 or R 12 and R 13 combine to form an optionally substituted heterocyclyl.
• the necrostatin is a Nec-4 related compound of Formula IX:
• Xi and X 2 are, independently, N or CR 4 ;
• X 3 is selected from O, S, NR 5 , or— (CR 5 ) 2 ;
• R 1 is selected from H, halogen, optionally substituted Ci-6alkyl, or optionally substituted Ci_6cycloalkyl, or optionally substituted aryl;
• R 2 is selected from H or optionally substituted Ci-6alkyl
• R 3 is optionally substituted aryl; each R 4 is selected from H, halogen, carboxamido, nitro, cyano, optionally substituted Ci_ 6 alkyl, or optionally substituted aryl;
• R 5 is selected from H, halogen, optionally substituted Ci- 6 alkyl, or optionally substituted aryl;
• each R 6 and R 7 is, independently, selected from H, optionally substituted Ci- 6 alkyl, or aryl;
• n 0, 1, 2, or 3.
• Xi and X 2 are N
• X3 is S
• Y is C(O)
• Z is CH 2
• R 2 is H
• R 3 is 2-chloro-6-fluoro-phenyl
• R 1 , R 2 , R 3 , R 6 and R 7 are as defined in Formula (IX).
• the necrostatin is a Nec-4 related compound selected from:
• Nec-4 related compounds described above can be prepared based on synthetic procedures described in the literature, such as U.S. Patent Application Publication No.
• cycloalkyls have from about 3 to about 10 carbon atoms in their ring structure, and alternatively about 5, 6 or 7 carbons in the ring structure.
• exemplary alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec- butyl, isobutyl, tert-butyl, cyclopropyl, and cyclobutyl.
• alkoxyl or "alkoxy” are art-recognized and refer to an alkyl group, as defined above, having an oxygen radical attached thereto.
• Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like.
• An "ether” is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as may be represented by one of -O-alkyl, -O-alkenyl, or -O-alkynyl.
• alkylene refers to a diradical of an alkyl group.
• An exemplary alkylene group is -CH 2 CH 2 -.
• aralkyl refers to an alkyl group substituted with an aryl group.
• heteroarylkyl refers to an alkyl group substituted with a heteroaryl group.
• alkenyl refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond, such as a straight or branched group of 2-12, 2-10, or 2-6 carbon atoms, referred to herein as C2-Ci2alkenyl, C2-Cioalkenyl, and C2-C 6 alkenyl, respectively.
• alkenyl groups include, but are not limited to, vinyl, allyl, butenyl, pentenyl, hexenyl, butadienyl, pentadienyl, hexadienyl, 2-ethylhexenyl, 2 -propy 1-2 -butenyl, 4- (2-methyl-3-butene)-pentenyl, etc.
• aryl groups include phenyl, naphthyl, anthracenyl, and the like. Unless specified otherwise, the aromatic ring may be substituted at one or more ring positions with, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, carboxylic acid, -C(0)alkyl, -CC ⁇ alkyl, carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamido, sulfonamide, ketone, aldehyde, ester, heterocyclyl, heteroaryl, -CF 3 , -CN, or the like.
• phenylene refers to a multivalent radical (e.g., a divalent or trivalent radical)
• a divalent valent radical of benzene is illustrated by the formula .
• heterocyclyl or “heterocyclic group” are art-recognized and refer to saturated, partially unsaturated, or aromatic 3- to 10-membered ring structures, alternatively 3- to 7-membered rings, whose ring structures include one to four heteroatoms, such as nitrogen, oxygen, and sulfur. Heterocycles may also be mono-, bi-, or other multi-cyclic ring systems. A heterocycle may be fused to one or more aryl, partially unsaturated, or saturated rings.
• Heterocyclyl groups include, for example, biotinyl, chromenyl, dihydrofuryl, dihydroindolyl, dihydropyranyl, dihydrothienyl, dithiazolyl, homopiperidinyl, imidazolidinyl, isoquinolyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, oxolanyl, oxazolidinyl, phenoxanthenyl, piperazinyl, piperidinyl, pyranyl, pyrazolidinyl, pyrazolinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolidin-2-onyl, pyrrolinyl, tetrahydrofuryl, tetrahydroisoquinolyl, tetrahydropyranyl, tetrahydroquinolyl, thiazolidinyl, th
• the heterocyclic ring is optionally substituted at one or more positions with substituents such as alkanoyl, alkoxy, alkyl, alkenyl, alkynyl, amido, amidino, amino, aryl, arylalkyl, azido, carbamate, carbonate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, imino, ketone, nitro, phosphate, phosphonato, phosphinato, sulfate, sulfide, sulfonamido, sulfonyl and thiocarbonyl.
• the heterocyclcyl group is not substituted, i.e., it is unsubstituted.
• heteroaryl is art-recognized and refers to aromatic groups that include at least one ring heteroatom. In certain instances, a heteroaryl group contains 1, 2, 3, or 4 ring heteroatoms. Representative examples of heteroaryl groups include pyrrolyl, furanyl, thiophenyl, imidazolyl, oxazolyl, thiazolyl, triazolyl, pyrazolyl, pyridinyl, pyrazinyl, pyridazinyl and pyrimidinyl, and the like.
• the heteroaryl ring may be substituted at one or more ring positions with, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, carboxylic acid, -C(0)alkyl, -CC ⁇ alkyl, carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamido, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aryl, -CF 3 , -CN, or the like.
• heteroaryl also includes polycyclic ring systems having two or more rings in which two or more carbons are common to two adjoining rings (the rings are "fused rings") wherein at least one of the rings is heteroaromatic, e.g., the other cyclic rings may be cycloalkyls,
• cycloalkenyls cycloalkynyls, and/or aryls.
• heteroarylene refers to a multi-valent (e.g., di-valent or trivalent) aromatic group that comprises at least one ring heteroatom.
• An exemplary “heteroarylene” is pyridinylene, which is a multi-valent ridine.
• a divalent radical of pyridine is illustrated by the formula
• ortho, meta and para are art-recognized and refer to 1,2-, 1,3- and 1,4- disubstituted benzenes, respectively.
• 1,2-dimethylbenzene and ortho- dimethylbenzene are synonymous.
• amine and “amino” are art-recognized and refer to both unsubstituted and substituted amines, e.g., a moiety that may be represented by the general formula:
• R 50 and R 51 each independently represent hydrogen, alkyl, alkenyl, or -(CH 2 ) m -R 61 ; or R 50 and R 51 , taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure; wherein R 61 is aryl, cycloalkyl, cycloalkenyl, a heterocycle or a poly cycle; and m is zero or an integer in the range of 1 to 8.
• R 50 and R 51 each independently represent hydrogen or alkyl.
• R a , R b and R c are each independently selected from alkoxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydrogen, hydroxyl, ketone, and nitro.
• the amide can be attached to another group through the carbon, the nitrogen, R b , R c , or R a .
• the amide also may be cyclic, for example R b and R c , R a and R b , or R a and R c may be joined to form a 3- to 12-membered ring, such as a 3- to 10- membered ring or a 5- to 6-membered ring.
• the term "carboxamido" refers to the structure -C(0)NR b Rc.
• sulfonamide or “sulfonamido” as used herein refers to a radical having the structure -N(R r )-S(0)2-R s - or -S(0)2-N(R r )R s , where R r , and R s can be, for example, hydrogen, alkyl, aryl, cycloalkyl, and heterocyclyl.
• Exemplary sulfonamides include alkylsulfonamides (e.g., where R s is alkyl), arylsulfonamides (e.g., where R s is aryl), cycloalkyl sulfonamides (e.g., where R s is cycloalkyl), and heterocyclyl sulfonamides (e.g., where R s is heterocyclyl), etc.
• sulfonyl refers to a radical having the structure R U S02-, where R u can be alkyl, aryl, cycloalkyl, and heterocyclyl, e.g., alkylsulfonyl.
• the term "optionally substituted” as used herein means that the specified group may be substituted at one, two or more positions with, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, carboxylic acid, -C(0)alkyl, -C0 2 alkyl, carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamido, sulfonamide, ketone, aldehyde, ester, heterocyclyl, heteroaryl, -CF 3 , -CN, or the like.
• the term "therapeutically effective amount” is understood to mean the amount of an active ingredient, for example, a necrostatin (e.g., necrostatin- 1 or necrostatin- 4) and/or a pan-caspase inhibitor (e.g., ZVAD or IDN-6556) that is sufficient to promote axon regeneration, preserve neuron viability, and/or promote nerve function in a CNS neuron.
• the compounds of the invention are administered in amounts effective at, e.g., promoting axon regeneration, preserving neuron viability, promoting nerve function, increasing efficacy compared to monotherapy with either drug alone, preserving or improving cognitive functions, preserving or improving sensory functions, and/or preserving or improving motor functions. It is understood that preserving cognitive, sensory, or motor functions, includes stabilizing these functions and/or slowing the decline of these functions.
• pharmaceutically acceptable or “pharmacologically acceptable” mean molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or to a human, as appropriate.
• pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
• the CNS neuron may be ex vivo.
• the CNS neuron may be isolated from a subject and maintained in an in vitro culture.
• the CNS neuron may be present in vivo.
• a method for promoting nerve function following injury to a CNS neuron comprises administering to a subject an effective amount of a necrosis inhibitor and an effective amount of an apoptosis inhibitor thereby to promote CNS neuron function.
• a method for preserving the viability of a CNS neuron comprising administering to the subject an effective amount of a necrosis inhibitor and an effective amount of an apoptosis inhibitor thereby to preserve the viability of the CNS neuron.
• the CNS neuron may be capable of supporting axonal regeneration.
• the invention provides a method of treating a CNS disorder in a subject in need thereof, wherein a symptom of the CNS disorder is axon degeneration or injury within a CNS neuron.
• the method comprises administering to the subject an effective amount of a necrosis inhibitor and an effective amount of an apoptosis inhibitor thereby to promote regeneration of an axon in a CNS neuron affected by the CNS disorder.
• neuron function may be measured, for example, as an indication of axon regeneration. It is also contemplated that, following administration of the necrosis inhibitor and the apoptosis inhibitor, the neuron function of the CNS neuron is preserved or improved relative to the neuron function prior to administration of the necrosis inhibitor and the apoptosis inhibitor.
• the CNS disorder affects a subject's movement and/or strength, such as injury to the brain or spinal cord, or a neurodegenerative CNS disorder such as Parkinson's disease, frontotemporal dementia, dementia with Lewy bodies, corticobasal degeneration, progress supranuclear palsy, Huntington's disease, multiple system atrophy, amyotropic lateral sclerosis, and hereditary spastic pararesis.
• a subject's movement and/or strength such as injury to the brain or spinal cord
• a neurodegenerative CNS disorder such as Parkinson's disease, frontotemporal dementia, dementia with Lewy bodies, corticobasal degeneration, progress supranuclear palsy, Huntington's disease, multiple system atrophy, amyotropic lateral sclerosis, and hereditary spastic pararesis.
• the invention provides a method of promoting axon regeneration in a CNS neuron, wherein the method comprises reducing the production and/or activity of a RIP-1 kinase and/or a RIP-3 kinase in the CNS neuron thereby promoting axon regeneration in a CNS neuron.
• the reduction in the production or activity of the RIP-1 kinase and/or the RIP-3 kinase can achieved by administering an effective amount of RIP kinase (RIPK) inhibitor, e.g., a necrostatin.
• RIPK RIP kinase
• the necrostatin can be administered to give a final concentration of greater than about 10 ⁇ , for example, in the range of about 10 ⁇ to about 1000 ⁇ .
• the final concentration refers to final concentration in, for example, the blood, the cerebrospinal fluid, or localized region of treatment (e.g., site of injury).
• the necrostatin can be administered in an amount sufficient to give a final concentration of necrostatin in an amount of greater than about 10 ⁇ .
• the necrostatin can be administered in an amount sufficient to give a final concentration of necrostatin in an amount of greater than about 10 ⁇ .
• the necrostatin can be administered in an amount sufficient to give a final concentration of necrostatin in an amount of greater than about 50 ⁇ .
• the necrostatin can be administered in an amount sufficient to give a final concentration of necrostatin in an amount of greater than about 100 ⁇ .
• the necrostatin may be administered in an amount sufficient to give a final concentration of necrostatin in an amount in the range from about 10 ⁇ to about 1000 ⁇ , 50 ⁇ to about 1000 ⁇ , 80 ⁇ to about 1000 ⁇ , about 100 ⁇ to about 1000 ⁇ , about 150 ⁇ to about 1000 ⁇ , from about 200 ⁇ to about 800 ⁇ , or from about 200 ⁇ to about 600 ⁇ .
• the necrostatin is administered in an amount sufficient to give a final concentration of necrostatin in an amount of about 400 ⁇ .
• the necrostatin can be administered in an amount sufficient to give a final concentration of necrostatin in an amount of greater than about 100 ⁇ .
• the apoptosis inhibitor can be administered in an amount sufficient to give a final concentration of the inhibitor in an amount in the range from about 3 ⁇ to about 500 ⁇ , from about 80 ⁇ to about 500 ⁇ , 100 ⁇ to about 500 ⁇ , 125 ⁇ to about 500 ⁇ , 150 ⁇ to about 500 ⁇ or from about 200 ⁇ to about 400 ⁇ .
• apoptosis inhibitor e.g., the pan-caspase inhibitor
• from about 0.025 mg to about 4 mg, from about 0.035 mg to about 2 mg, from about 0.05 mg to about 2 mg, from about 0.1 mg to about 2 mg, from about 0.2 mg to about 1 mg, or from about 0.2 mg to about 0.8 mg of the necrosis inhibitor (e.g., a necrostatin) can be administered.
• the necrosis inhibitor e.g., a necrostatin
• from about 0.05 mg to about 2 mg, from about 0.2 mg to about 2 mg, from about 0.05 mg to about 1.5 mg, from about 0.15 mg to about 1.5 mg, from about 0.4 mg to about 1 mg, or from about 0.5 mg to about 0.8 mg of an apoptosis inhibitor (e.g., a pan-caspase inhibitor, e.g., ZVAD) can be administered.
• an apoptosis inhibitor e.g., a pan-caspase inhibitor, e.g., ZVAD
• one or more of a necrosis inhibitor, one or more of an apoptosis inhibitor, or one or more of a necrosis inhibitor and one or more of an apoptosis inhibitor can be administered in amounts sufficient to preserve the viability and/or promote axon regeneration and/or nerve function of an affected CNS neuron.
• the necrosis inhibitor is a necrostatin, for example, necrostatin- 1 , a necrostatin-2, a necrostatin-4, a necrostatin-5, and a necrostatin-7.
• a necrostatin for example, necrostatin- 1 , a necrostatin-2, a necrostatin-4, a necrostatin-5, and a necrostatin-7.
• a necrostatin for example, necrostatin- 1 , a necrostatin-2, a necrostatin-4, a necrostatin-5, and a necrostatin-7.
• apoptosis inhibitors e.g., IDN-6556
• one or more of the necrostatins shown by Formua I, I-A, I-B, I-C, I-D, I-E, I-F, I-G, II, II-A, III, IV, IV-A, IV-B, V, V-A, VII, VIII, VIII-A, IX, or IX-A can be administered with one or more of the apoptosis inhibitors (e.g., IDN-6556 or IDN-6734) listed below.
• the apoptosis inhibitors e.g., IDN-6556 or IDN-6734
• the necrosis inhibitor reduces the production and/or activity of a RIP-1 kinase and/or a RIP-3 kinase.
• RIP kinase inhibitors e.g., RIP-1 kinase and/or RIP-3 kinase inhibitors
• RNAs including small inhibitory RNAs (siRNAs) and short hairpin RNAs (shRNAs). Methods for designing and synthesizing siRNAs and shRNAs are well known in the art.
• Exemplary RIP- 1 kinase inhibitors include, for example, a pSIREN-RIP-1 shRNA construct which targets RIP-1 kinase as disclosed in Kaiser et al, (2008) JOURNAL OF IMMUNOLOGY 181 :6427-6434.
• Exemplary RIP-3 kinase inhibitors include, for example, sc-61482-SH and sc-135170 available from Santa Cruz Biotechnology.
• RIP kinase inhibitors e.g., RIP-1 kinase and/or RIP-3 kinase inhibitors
• IAPs inhibitor of apoptosis proteins
• the one or more apoptosis inhibitors may include a pan- caspase inhibitor.
• the pan-caspase inhibitor can be ZVAD (i.e., Z-Val-Ala-Asp(OMe)- CH2F*), IDN-6556 available from Conatus Pharmaceuticals (i.e., (3- ⁇ 2-[(2-tert-butyl- phenylaminooxalyl)-amino]-propionylamino ⁇ -4-oxo-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoic acid) (3- ⁇ 2-[(2-tert-butyl-phenylaminooxalyl)-amino]-propionylamino ⁇ -4-oxo-5-(2,3,5,6- tetrafluoro-phenoxy)-pentanoic acid), IDN-6734 available from Conatus Pharmaceuticals, VX- 799 available from Vertex Pharmaceuticals, M
• the pan-caspase inhibitor can be a cocktail of caspase inhibitors including two or more specific caspase inhibitors (e.g., synthetic caspase inhibitors) such as a caspase 1 inhibitor, a caspase 2 inhibitor, a caspase 3 inhibitor, a caspase 4 inhibitor, a caspase 5 inhibitor, a caspase 6 inhibitor, a caspase 7 inhibitor, a caspase 8 inhibitor, and a caspase 9 inhibitor. It is contemplated that one or more of the pan-caspase inhibitors may be used in combination with one or more necrostatins (e.g., necrostain-1 and/or necrostatin-4).
• necrostatins e.g., necrostain-1 and/or necrostatin-4
• Exemplary synthetic caspase 1 inhibitors include, for example, Ac-N-Me-Tyr-Val- Ala-Asp-aldehyde (SEQ ID NO: 7), Ac-Trp-Glu-His-Asp-aldehyde (SEQ ID NO: 8), Ac-Tyr- N-Me-Val-Ala-N-Me-Asp-aldehyde (SEQ ID NO: 9), Ac-Tyr-Val-Ala-Asp-Aldehyde (SEQ ID NO: 10), Ac-Tyr-Val-Ala-Asp-chloromethylketone (SEQ ID NO: 1 1), Ac-Tyr-Val-Ala-Asp- 2,6-dimethylbenzoyloxymethylketone (SEQ ID NO: 12), Ac-Tyr-Val-Ala-Asp(OtBu)- aldehyde-dimethyl acetol (SEQ ID NO: 13),
• exemplary caspase 1 inhibitors include, for example, Z-Val-Ala-Asp- fluoromethylketone, biotin-X-Val-Ala-Asp-fluoromethylketone, Ac-Val-Ala-Asp-aldehyde, Boc-Asp-fluoromethylketone, Ac-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu- Leu-Pro-Tyr-Val-Ala-Asp-aldehyde (SEQ ID NO: 1), biotin-Tyr-Val-Ala-Asp- fluoroacyloxymethylketone (SEQ ID NO: 20), Ac-Tyr-Val-Ala-Asp-acyloxymethylketone (SEQ ID NO: 21), Z-Asp-CH2-DCB, and Z-Tyr-Val-Ala-Asp-fluoromethylketone (SEQ ID NO:
• Exemplary synthetic caspase 2 inhibitors include, for example, Ac-Val-Asp-Val- Ala-Asp-aldehyde (SEQ ID NO: 23), which can be obtained from Bachem Bioscience Inc., PA, and Z-Val-Asp-Val-Ala-Asp-fluoromethylketone (SEQ ID NO: 24), which can be obtained from Calbiochem, CA.
• SEQ ID NO: 23 Ac-Val-Asp-Val- Ala-Asp-aldehyde
• SEQ ID NO: 24 Z-Val-Asp-Val-Ala-Asp-fluoromethylketone
• Exemplary synthetic caspase 3 precursor protease inhibitors include, for example, Ac-Glu-Ser-Met-Asp-aldehyde (pseudo acid) (SEQ ID NO: 25) and Ac-Ile-Glu-Thr-Asp- aldehyde (pseudo acid) (SEQ ID NO: 26) which can be obtained from Bachem Bioscience Inc., PA.
• Exemplary synthetic caspase 3 inhibitors include, for example, Ac-Asp-Glu-Val-Asp- aldehyde (SEQ ID NO: 27), Ac-Asp-Met-Gln-Asp-aldehyde (SEQ ID NO: 28), biotinyl-Asp- Glu-Val-Asp-aldehyde (SEQ ID NO: 29), Z-Asp-Glu-Val-Asp-chloromethylketone (SEQ ID NO: 30), Z-Asp(OMe)-Glu(OMe)-Val-DL-Asp(OMe)-fluoromethylketone (SEQ ID NO: 31), and Z-Val-Ala-DL-Asp(OMe)-fluoromethylketone which can be obtained from Bachem Bioscience Inc., PA.
• caspase 3 inhibitors include, for example, Ac-Ala-Ala- Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Asp-Glu-Val-Asp-aldehyde (SEQ ID NO: 2), biotin-X-Asp-Glu-Val-Asp-fluoromethylketone (SEQ ID NO: 32), Ac-Asp- Glu-Val-Asp-chloromethylketone (SEQ ID NO: 33), all of which are available from
• Another exemplary caspase 3 inhibitor includes, the caspase 3 inhibitor N- benzyloxycarbonal-Asp(OMe)-Glu(OMe)-Val-Asp(Ome)-fluoromethyketone (z- Asp-Glu-Val- Asp-fmk) (SEQ ID NO: 34), which is available from Enzyme Systems Products.
• Additional exemplary caspase 3 inhibitors include M-826 and M-791 available from Merck-Frosst, Immunocasp-3, Ad-G/iCasp3, and PEF-F8-CP3.
• Exemplary synthetic caspase 4 inhibitors include, for example, Ac-Leu-Glu-Val- Asp-aldehyde (SEQ ID NO: 35) and Z-Tyr-Val-Ala-DL-Asp-fluoromethylketone (SEQ ID NO: 36), which can be obtained from Bachem Bioscience Inc., PA, and Ac-Ala-Ala-Val-Ala-Leu- Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Leu-Glu-Val-Pro-aldehyde (SEQ ID NO: 3), which can be obtained from Calbiochem, CA.
• Exemplary synthetic caspase 5 inhibitors include, for example, Z-Trp-His-Glu-Asp- fluoromethylketone (SEQ ID NO: 37), which can be obtained from Calbiochem, CA, and Ac- Trp-Glu-His-Asp-aldehyde (SEQ ID NO: 38) and Z-Trp-Glu(0-Me)-His-Asp(0-Me) fluoromethylketone (SEQ ID NO: 39), which can be obtained from Sigma Aldrich, Germany.
• Exemplary synthetic caspase 6 inhibitors include, for example, Ac-Val-Glu-Ile-Asp- aldehyde (SEQ ID NO: 40), Z-Val-Glu-Ile-Asp-fluoromethylketone (SEQ ID NO: 41), and Ac- Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Val-Glu-Ile-Asp- aldehyde (SEQ ID NO: 4), which can be obtained from Calbiochem.
• Another exemplary caspase 6 inhibitor includes Immunocasp-6.
• Exemplary synthetic caspase 7 inhibitors include, for example, Z-Asp(OMe)-Gln- Met-Asp(OMe) fluoromethylketone (SEQ ID NO: 42), Ac-Asp-Glu-Val-Asp-aldehyde (SEQ ID NO: 43), Biotin-Asp-Glu-Val-Asp-fluoromethylketone (SEQ ID NO: 44), Z-Asp-Glu-Val- Asp-fluoromethylketone (SEQ ID NO: 45), Ac-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu- Leu-Ala-Leu-Leu-Ala-Pro-Asp-Glu-Val-Asp-aldehyde (SEQ ID NO: 2), which can be obtained from Sigma Aldrich, Germany.
• Exemplary synthetic caspase 8 inhibitors include, for example, Ac-Asp-Glu-Val- Asp-aldehyde (SEQ ID NO: 46), Ac-Ile-Glu-Pro-Asp-aldehyde (SEQ ID NO: 47), Ac-Ile-Glu- Thr-Asp-aldehyde (SEQ ID NO: 48), Ac-Trp-Glu-His-Asp-aldehyde (SEQ ID NO: 49) and Boc-Ala-Glu-Val-Asp-aldehyde (SEQ ID NO: 50) which can be obtained from Bachem
• caspase 8 inhibitors include, for example, Ac-Ala-Ala- Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Ile-Glu-Thr- Asp-aldehyde (SEQ ID NO: 5) and Z-Ile-Glu-Thr-Asp-fluoromethylketone (SEQ ID NO: 51), which can be obtained from Calbiochem, CA.
• Exemplary synthetic caspase 9 inhibitors include, for example, Ac-Asp-Glu-Val- Asp-aldehyde (SEQ ID NO: 52), Ac-Leu-Glu-His-Asp-aldehyde (SEQ ID NO: 53), and Ac- Leu-Glu-His-Asp-chloromethylketone (SEQ ID NO: 54) which can be obtained from Bachem Bioscience Inc., PA.
• exemplary caspase 9 inhibitors include, for example, Z-Leu-Glu- His-Asp-fluoromethylketone (SEQ ID NO: 55) and Ac-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala- Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Leu-Glu-His-Asp-aldehyde (SEQ ID NO:6), which can be obtained from Calbiochem, CA.
• Another exemplary caspase 9 inhibitor includes
• FKBP12/caspase-9 fusion protein FKBP12/caspase-9 fusion protein.
• the pan-caspase inhibitor may also be an endogenous caspase inhibitor or a combination of an endogenous caspase inhibitor with one or more synthetic caspase inhibitors.
• one useful class of endogenous caspase inhibitor includes proteins known as inhibitors of apoptosis proteins (IAPs) (Deveraux et al, (1998) EMBO J. 17(8): 2215-2223) including bioactive fragments and analogs thereof.
• IAPs X-linked inhibitor of apoptosis protein
• Another exemplary IAP includes survivin (see, U.S. Patent No.
• the one or more apoptosis inhibitors may target the inhibitor of apoptosis proteins (IAPs) and second mitochondria-derived activator of caspases (SMACs).
• IAPs apoptosis proteins
• SMACs second mitochondria-derived activator of caspases
• Exemplary apoptosis inhibitors that target IAPs and SMACs include, for example, BIR3 antagonists available from Idun Pharmaceuticals, capped tripeptide XIAP antagonists from Abbot Laboratories, TWX024, polyphenylurea derivatives, SMAC-mimetic compounds, embelin, XIAP antisense and RNAi constructs, AEG35156/GEM ® 640 available from Aegera Therapeutics, HIV-Tat- and polyarginine conjugated SMAC peptides, and nonpeptide small- molecule SMAC mimetics. It is contemplated that one or more of the apoptosis inhibitors which target IAPs and SMACs may
• the one or more apoptosis inhibitors may target the TNF- related apoptosis-inducing ligand (TRAIL) receptors.
• TRAIL TNF-related apoptosis-inducing ligand
• Exemplary apoptosis inhibitors that target the TRAIL receptors include, for example, HGS-ETR1, HGS-ETR2, and HGS-TR2J available from Human Genome Sciences, and PRO 1762 available from Amgen. It is contemplated that one or more of the apoptosis inhibitors which target the TRAIL receptors may be used in combination with one or more necrostatins (e.g., necrostain-1 and/or necrostatin-4).
• necrostatins e.g., necrostain-1 and/or necrostatin-4.
• the one or more apoptosis inhibitors may target the tumor necrosis factor (TNF).
• TNF tumor necrosis factor
• exemplary apoptosis inhibitors that target TNF include, for example, recombinant TNF-a, adalimumab available from Abbott, infliximab available from Centocor Ortho Biotech Inc., etanercept from Amgen, CDP571 available from Celltech, and ISIS 104838 (a 2'-0-methoxyethyl antisense construct against TNF-alpha) available from ISIS
• the one or more apoptosis inhibitors may target the Bcl-2 proteins.
• Exemplary apoptosis inhibitors that target the Bcl-2 proteins include, for example, Bcl-2 blockers available from Idun Pharmaceuticals and Abbot Laboratories, GxOl series of compounds available from Gemin X Pharmaceuticals, Bcl-2 small-molecule antagonist, Tetrocarcin-A derivatives available from Kyowa Hakko Kogyo Co., Chelerythrine, antimycin A derivatives, HA 14-1, synthetic compound binding to the BH3 of Bcl-2, Genasense available from Sanofi-Aventis, ISIS 22783 available from ISIS Pharmaceuticals, bispecific Bcl-2/Bcl-XL antisense, BH3 peptides from Bax, Bak, Bid or Bad, SAHBs, and BH3Is. It is contemplated that one or more of the apoptosis inhibitors which target the Bcl-2 proteins may be
• the one or more apoptosis inhibitors may target p53.
• Exemplary apoptosis inhibitors that target p53 include, for example, INGN201 available from Invitrogen Therapeutics, SCH58500 available from Schering-Plough, ONYX-015 available from Onyx Pharmaceuticals, C-terminal p53 peptides, CDB3, Amifostine, CP31398 available from Pfizer, Prima- 1, HPF E6-binding peptide aptamers, Nutlins available from Roche, Chalcones, Small peptide compounds, and Pifithrin-a. It is contemplated that one or more of the apoptosis inhibitors which target p53 may be used in combination with one or more necrostatins (e.g., necrostain-1 and/or necrostatin-4).
• necrostatins e.g., necrostain-1 and/or necrostatin-4.
• necrostatins may be used in combination with a pan-caspase inhibitor.
• necrostain-1 and/or necrostatin-4 may be used in combination with ZVAD available from R&D Systems (Cat. No. FMKOOl) and Promega (Cat. No. G7231).
• necrostain-1 and/or necrostatin-4 may be used in combination with IDN-6556 available from Conatus Pharmaceuticals.
• necrostain-1 and/or necrostatin-4 may be used in combination with IDN-6734 available from Conatus Pharmaceuticals.
• necrostatins may be used in combination with a TNF inhibitor.
• necrostain-1 and/or necrostatin-4 may be used in combination with adalimumab available from Abbot Laboratories.
• necrostain-1 and/or necrostatin-4 may be used in combination with etanercept available from Amgen, Inc.
• necrostain-1 and/or necrostatin-4 may be used in combination with infiximab available from Centocor Ortho Biotech, Inc.
• necrostatins may be used in combination with a p53 agonist.
• necrostain-1 and/or necrostatin-4 may be used in combination with INGN 201 available from Invitrogen Therapeutics.
• necrostain-1 and/or necrostatin-4 may be used in combination with nutlins, for example, nutlin-3 available from Cayman Chemical (Cat. No. 10004372).
• necrostain-1 and/or necrostatin-4 may be used in combination with CP31398 available from Tocris Bioscience (Cat. No. 3023).
• apoptotic inhibitor can modulate both the apoptotic and necrotic pathways, and depending upon the specific necrosis inhibitor chosen, it is possible that the necrosis inhibitor can modulate both the necrotic and apoptotic pathways.
• a RIP-1 inhibitor may inhibit both necrotic and apoptotic cell death thus preserving the viability of CNS neurons and promoting axon regeneration in a subject with a CNS disorder as disclosed herein.
• the disclosed methods promote axon regeneration of a CNS neuron. Further, the disclosed methods preserve neuron viability and/or promote nerve function following injury to a CNS neuron. Assessment of axonal regeneration and nerve function may be monitored by, functional tests which are well-established in the art, such as, for example, magnetic resonance imaging (MRI) and tests involving evaluations of a subject's cognitive, motor, and sensory functions.
• MRI magnetic resonance imaging
• evoked potential also called evoked response
• neurological computed tomography also known as a neurological CT scan
• brain damage may be performed to monitor recovery from brain damage.
• axonal regeneration in a stroke patient may be measured by the NIH Stroke Scale ( IHSS).
• IHSS NIH Stroke Scale
• the NIHSS is a standardized neurological examination that measures several aspects of brain function, including consciousness, vision, sensation, movement, speech, and language, and is intended describe the neurological deficits found in stroke patients.
• Other functional tests may be based on, for example, the Barthel Index (BI), which measures self-care and mobility.
• BI Barthel Index
• the BI assesses a subject's ability to perform tasks such as personal toileting, feeding, mobility from bed to chair, transfers, and bathing.
• Functional test may also include the Modified Rankin Scale (mRS), which is commonly used for measuring the degree of disability or dependence in the daily activities of stroke patients.
• mRS Modified Rankin Scale
• necrosis inhibitor and the apoptosis inhibitor may be administered directly to the site of injury or systemically, e.g., by oral or parenteral routes.
• Parenteral routes include, for example, intravenous, intrarterial, intracranial, intraorbital, opthalmalic, intraventricular, intraspinal (e.g., into the cerebrospinal fluid), intracistemal, intramuscular, intradermal, subcutaneous, intranasal and intraperitoneal routes. It is contemplated that local modes of administration may reduce or eliminate the incidence of potential side effects (e.g., systemic toxicity) that may occur during systemic administration.
• side effects e.g., systemic toxicity
• the necrosis inhibitor and the apoptosis inhibitor may be administered to a subject simultaneously or sequentially. It will be appreciated that when administered simultaneously, the necrosis inhibitor and the apoptosis inhibitor may be in the same pharmaceutically acceptable carrier or the two drugs may be dissolved or dispersed in separate pharmaceutical carriers, which are administered at the same time. Alternatively, the drugs may be provided in separate dosage forms and administered sequentially.
• the necrostatin may be administered before the pan-caspase inhibitor. In other examples, the pan- caspase inhibitor may be administered before the necrostatin.
• a single active agent may inhibit both necrosis and apoptosis.
• the necrosis inhibitor and/or the apoptosis inhibitor may be solubilized in a pharmaceutically acceptable carrier.
• One or both inhibitors also may be administered in a pharmaceutically acceptable carrier or vehicle so that administration does not otherwise adversely affect the recipient's electrolyte and/or volume balance.
• the carrier may comprise, for example, physiologic saline or other buffer system.
• the necrostatin, the pan-caspase inhibitor, or both the necrostatin and the pan-caspase inhibitor may be solubilized in PBS or another aqueous buffer by sonication.
• one or both drugs may be solubilized using conventional solvent or solubilization systems, for example, dimethyl sulfoxide (DMSO), dimethoxyethane (DME), dimethylformamide (DMF), cyclodextran, micelles, liposomes, liposomal agents, and other solvents known in the art to aid in the solubilization and administration of hydrophobic agents.
• DMSO dimethyl sulfoxide
• DME dimethoxyethane
• DMF dimethylformamide
• cyclodextran cyclodextran
• micelles micelles
• liposomes liposomal agents
• liposomal agents and other solvents known in the art to aid in the solubilization and administration of hydrophobic agents.
• the necrosis inhibitor and/or the apoptosis inhibitor may be solubilized in a liposome or microsphere.
• Methods for delivery of a drug or combination of drugs in liposomes and/or microspheres are well-known in the art.
• the necrosis inhibitor and/or the apoptosis inhibitor may be formulated so as to permit release of one or both inhibitors over a prolonged period of time.
• a release system can include a matrix of a biodegradable material or a material, which releases the incorporated active agents.
• the active agents can be homogeneously or heterogeneous ly distributed within a release system.
• release systems may be useful in the practice of the invention, however, the choice of the appropriate system will depend upon the rate of release required by a particular drug regime. Both non-degradable and degradable release systems can be used.
• Suitable release systems include polymers and polymeric matrices, non-polymeric matrices, or inorganic and organic excipients and diluents such as, but not limited to, calcium carbonate and sugar (for example, trehalose). Release systems may be natural or synthetic. However, under certain circumstances, synthetic release systems are preferred because generally they are more reliable, more reproducible and produce more defined release profiles. The release system material can be selected so that inhibitors having different molecular weights are released by diffusion through or degradation of the material.
• Representative synthetic, biodegradable polymers include, for example: polyamides such as poly(amino acids) and poly(peptides); polyesters such as poly(lactic acid),
• Intravitreal injections were performed as follows. Briefly, the tip of a 33 gauge needle (Hamilton, Reno, NV) was carefully inserted through the sclera into the intravitreal space to reduce intraocular pressure. Then, the needle was extracted, loaded with compounds and tangentially reinserted through the sclera into the intravitreal space, inducing a self-sealing wound tunnel. After injection, the absence of choroidal bleeding was confirmed. At specified times after injury, mice were sacrificed with an overdose of sodium pentobarbital, and eyes were enucleated.
• the optic nerve cannot regenerate if injured, leaving victims of traumatic nerve injury or degenerative diseases such as glaucoma with life-long visual losses. This situation can be, at least, partially reversed by enhancing the intrinsic growth state of retinal ganglion cells (RGCs).
• RGCs retinal ganglion cells
• the efficacy of necrosis inhibitor and a pan-caspase inhibitor in promoting RGC survival and axon regeneration is investigated using a mouse optic nerve crush model.
• a Necrosis Inhibitor in Combination with a Caspase Inhibitor Promotes RGC
• mice were subjected to optic nerve crush surgery. Specifically, animals were anesthetized with an intraperitoneal injection of ketamine (60-80 mg/kg: Phoenix
• xylazine (10-15 mg/kg: Bayer, Shawnee Mission, KA). Animals were positioned in a stereotaxic apparatus and a 1-1.5 cm incision was made in the skin above the right orbit. Under microscopic illumination, the lachrymal glands and extraocular muscles were resected to expose 3-4 mm of the optic nerve. The epineurium was slit open along the long axis, and the nerve was crushed 2 mm behind the eye with angled jeweler's forceps (Dumont # 5) for 10 seconds, avoiding injury to the ophthalmic artery. Nerve injury was verified by the appearance of a clearing at the crush site, while the vascular integrity of the retina was evaluated by fundoscopic examination. Cases in which the vascular integrity of the retina was in question were excluded from the study.
• mice were divided into four groups for treatment: vehicle group, ZVAD group (300 ⁇ ; given at day 0, day 3 and day 7 after injury), Nec-1 group (4 mM; given at day 0, day 3 and day 7 after injury), and ZVAD plus Nec-1 group (300 ⁇ and 4 mM, respectively; given either once or at day 0, day 3 and day 7 after injury).
• ZVAD group 300 ⁇ ; given at day 0, day 3 and day 7 after injury
• Nec-1 group 4 mM; given at day 0, day 3 and day 7 after injury
• ZVAD plus Nec-1 group 300 ⁇ and 4 mM, respectively; given either once or at day 0, day 3 and day 7 after injury.
• Zymosan 12.5 ⁇ g/ ⁇ l
• yeast cell wall preparation known to stimulate axonal regeneration.
• mice were subjected to optic nerve crush surgery as previously described. Subsequently, injured mice were divided into five groups of treatment: vehicle group, ZVAD group (300 ⁇ ; given at day 0, day 3 and day 7 after injury), Nec-1 group (4 mM; given at day 0, day 3 and day 7 after injury), ZVAD plus Nec-1 group (300 ⁇ and 4 mM, respectively; given once at day 0), and ZVAD plus Nec-1 group (300 ⁇ and 4 mM, respectively; given at day 0, day 3 and day 7 after injury).
• ZVAD group 300 ⁇ ; given at day 0, day 3 and day 7 after injury
• Nec-1 group 4 mM; given at day 0, day 3 and day 7 after injury
• ZVAD plus Nec-1 group 300 ⁇ and 4 mM, respectively; given once at day 0
• ZVAD plus Nec-1 group 300 ⁇ and 4 mM, respectively; given at day 0, day 3 and day 7 after injury.
• Axon regeneration was assessed by obtaining longitudinal sections of the optic nerve and counting the number of axons at pre-specified distances from the injury site.
• Figures 3A-3E show longitudinal sections of the optic nerve following optic nerve crush injury. The sections are stained with an antibody against ⁇ -tubulin, which marks axon fibers. In each photograph, an arrow indicates the sites of optic nerve injury, and staining beyond the injury site starting from left to right indicates axon regeneration (e.g., axons regenerate from the site of injury into the nerve).
• the specificity of motor axon regeneration can be investigated in the rat femoral nerve.
• axons that contribute to both cutaneous and muscle branches intermingle throughout the nerve.
• these axons regenerate, they have equal access to neighboring motor and sensory Schwann cell tubes in the distal nerve stump. This assures an element of "choice" at the axonal level.
• axons are segregated into terminal cutaneous and muscle branches. Motor axons are normally found only in the muscle branch, so any motor reinnervation of the cutaneous branch represents a pathfinding failure.
• HRP horseradish peroxidase
• FG fluoro-gold
• rats are divided into four treatment groups: vehicle group, ZVAD group, Nec-1 group, and ZVAD plus Nec-1 group. These agents are pumped onto the repair site, using an Alzet osmotic pump for at least 2 weeks. The outlet of the pump is sewn to muscle adjacent to the nerve repair, so that the nerve wound is continuously bathed with the necrosis inhibitor and the apoptosis inhibitor. Reinnervation of the distal femoral cutaneous and muscle branches can be quantified with tracers as described above.
• mice treated with ZVAD plus Nec- 1 will show an increase in the mean number of correct projections and a reduction in the mean number of incorrect projections to skin relative to a controls.
• Example 3 Efficacy of a Necrosis Inhibitor and a Pan-Caspase Inhibitor in a Rat Model of Damaged Vertebra
• Two-month old Sprague-Dawley rats (200-220 g) are used. Starting from 14 days before surgical operations, the animals undergo basic walking training for the Basso, Beattie and Bresnahan (BBB) test and the grid walk test, which measure locomotor functions. At 3 days before surgical operations, the animals are subjected to basic evaluations with respect to their behaviors and movement functions.
• BBB Basso, Beattie and Bresnahan
• Rats are anesthetized with 2 kg/ml of a mixture of 25 mg/ml of ketamine and 1.3 mg/ml of Rompun and subjected to L2 Ventral Laminectomy.
• the animals are intramuscularly injected with the antibiotic Cefalexin (5 mg/100 g bodyweight/day) to prevent infections.
• Spinal cord injury is induced by opening the second lumbar vertebra of each rat and puncturing a small hole (1 mm 2 ) in the outside of the left arcus vertebra using a microrongeur.
• the blade of a blade holder is inserted into the hole and knifed via the dura mater to the outside of the right arcus vertebra, thus causing traumatic damage at the abdominal portion of the spine.
• the dorsal musculature of the damaged spinal nerve portion is sutured and ligated with surgical clips. After the surgical operation, the rats are placed on warm sawdust to maintain their body temperature, and the portion below the abdominal region is massaged 3-4 times every day for 7 days so as to discharge the content of the bladder, until the autonomic bladder control thereof is completely restored.
• the rats are divided into four treatment groups: vehicle group, ZVAD group (given at day 0, day 3 and day 7 after injury), Nec-1 group (given at day 0, day 3 and day 7 after injury), and ZVAD plus Nec-1 group (given at day 0, day 3 and day 7 after injury).
• the rats are subjected to functional tests, such as the BBB test or the grid walk test, which measure their open-field walking ability and motor functions.
• the rats are further subjected to tests such as the footprint analysis,

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• 2012
  • 2012-10-22 JP JP2014537360A patent/JP6254090B2/ja active Active
  • 2012-10-22 HK HK15102094.4A patent/HK1203044A1/xx unknown
  • 2012-10-22 WO PCT/US2012/061324 patent/WO2013059791A2/en active Application Filing
  • 2012-10-22 CA CA2888805A patent/CA2888805C/en not_active Expired - Fee Related
  • 2012-10-22 EP EP12780381.5A patent/EP2773341A2/en not_active Withdrawn
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• 2015
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• 2017
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• 2018
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• 2019
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